Molecular Identification, Expression And Localization Analysis Of Odorant Receptor Genes Or1,Or2and Or3from Apis Cerana Cerana

Insects are the most diverse group animals on earth, the important thing for them is to maximize adapt their respective habitats. One such fundamental adaptation is the ability to detect and respond to chemical cues in the environment via a chemical sensor. The sophisticated olfactory system of insects is able to sense volatile odorants derived from food sources, host plants, oviposition sites, communication and mating. Researches on the olfactory molecular mechanism of insects are in favour of explore their biological behavior and explain the mechanism of co-evolution with their host plants, provide the basis for utilize economic insects efficiently and biological prevention and control agricultural insects. In this study, gene clone, sequence analysis, mRNA expression and cellular localization were researched on three odorant receptors from A. c. cerana, one of a precious honeybee resource of our country. The primary results are as follows:(1) The full-length cDNA of three odorant receptor genes, AcerOrl, AcerOr2and AcerOr3were cloned from the antennae of A. c. cerana by RT-PCR and RACE-PCR method. The cDNA sequences are1507,1763,1320bp and the GenBank Accession number are N792580, JN792581and JX049410. Their cDNA open reading frame encoded404,478,402amino acid residues with the molecular weight of about50kDa. The predicted proteins are hydrophilic with no signal peptide. AcerOr2includes seven putative transmembrane domains and AcerOrl and AcerOr3contain six ones. These traits are similar to typical characteristic of insects ORs.(2) We designed primers across introns based on the acquired cDNA sequences to clone the total introns of AcerOrl and AcerOr3with the GenBank Accession Number are JN544932and JX258126. The lengths from the transcriptional start site to polyadenylation signal are2049and1872bp. The sequence structures of AcerOr1and AcerOr3genes are very similar, both including five exons and four introns and own high level conservation in their sequences with77.3%identity on the DNA level and82.7%identity on the protein level. AcerOrl and AcerOr3are speculating the neighboring duplicated genes tandemly arrayed in the genome.(3) Using the BLAST tool, several homologous amino acid sequences to each gene were searched from Hymenopteran insect species. Multiple sequence alignments revealed that AcerOr2are fairly conserved and shares high levels of identity with other Orco orthologues (>75%identity). The identities between AcerOrl, AcerOr3and other homologous sequences are51.7%-98.2%and it is hardly to find out homolog in other order insects. The average dN/dS ratio in AcerOr2is0.031and in AcerOrl, AcerOr3is0.115. The results indicates that the3genes all have been undergone purifying selection and they may play important roles in the olfactory recognition process for honeybees. In addition, the results reflect that Or1and Or3are evolving more rapidly than the Orco gene.(4) Expression profiles of mRNA at different developmental stages in antennae of these3genes were examined by QRT-PCR. The results reveal that the AcerOrl, AcerOr2, AcerOr3transcripts are expressed both in worker and drone antennae. The expression levels of AcerOr2and AcerOr3are higher obviously in drone than in worker. Furthermore, the temporal expression patterns of the3genes are similar, that is transcripts are weakly expressed in larval and pupal stages and increase gradually along with ontogeny of bees. In workers, the expression levels are relative high at pupal period for every genes and reach their highest level around the eclosion time.(5) To investigate the cellular localization of AcerOrl, AcerOr2, AcerOr3mRNA in antennal, in situ hybridization was performed with digoxigenin-labelled probes. Hybridization signals reveal that the3odorant receptors are expressed in neuronal cells of sensilla trihodea and sensilla placodea on the basal of antennae. The labelling of AcerOr2mRNA is abundant, which is accordance with the result of QRT-PCR. AcerOrl and AcerOr3are less to be detected.In conclusion, we systematically studied the odorant receptor genes of AcerOr1, AcerOr2, AcerOr3from different profiles. It provides an important scientific basis for investigating the olfactory sensory mechanism, as well as provides new ideas for revealing the sensitive olfactory ability of A. c. cerana.